Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: NEUTRAL RED RELEASE METHOD
Principles of method if other than guideline:
PREDISAFE is a cell kit based on the use of a rabbit fibroblastic tell line (SIRC line)6 using cytotoxicity as endpoint. The test product is applied, for a short time, on the confluent monolayer of cells already preloaded with neutral red. Then, the variation in the viability between control cells and treated tells is measured according to the neutral red release (NRR) assay described by Borenfreund et al. (1984)
Different concentrations of the test product are used in the assay in order to determine the
amount inducing a 50% réduction in the NRR (IC50, inhibition concentration 50).
An in vitro/in vivo correlation matrix between the IC50 determined in the PREDISAFE assay
and the Draize irritation data, allows the assessment of the ocular tolerance of the test product (Guyomard et al., 1994)
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Tested: at the use concentration of 100% and at 3 lowers (10%, 30% and 50%) prepared from the use concentration (all dilutions were prepared in lipophilic solvent).

Test animals / tissue source

Species:
rabbit
Details on test animals or tissues and environmental conditions:
Confluent monolayer fibroblast cells isolated from rabbit cornea, and seeded in 24-well plates. Cells were receipt in an appropriate culture medium (called
shipping medium) to limit cellular metabolism during shipping.

Test system

Vehicle:
other: paraffin oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
4 concentrations were tested : 100% and at 3 lowers (10%, 30% and 50%)
Duration of treatment / exposure:
1 minute
Number of animals or in vitro replicates:
24-well plate
Details on study design:
Four hundred microlitres of fresh culture medium containing neutral red was added into each well
Fibroblasts were then incubated for 3 hours at 37°C in absence of CO2.
Following neutral red incorporation, cells were rinsed and test product was applied for 1 minute precisely.
In parallel, quality controls (the three reference products) and control (cells treated with the solvent hydrophilic and/or lipophilic) were performed.

After the short contact period, test product was removed and the cells were washed several times with the rinsing medium.
Five hundred microlitres of the lysis solution were added into each well and allowed shaking for few minutes.
After making the blank correction with the lysis solution, optical density was measured at 540 mn using a microplate reader.

Results and discussion

In vitro

Results
Irritation parameter:
other: Predisafe IC50
Remarks:
The concentration of test product inducing a 50% reduction of neutral red release (IC50) was determined graphically from the dose-response curve.
Value:
>= 50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

DRAIZE MAS

CLASSIFICATION

PREDISAFETMIC50

 <15

 Class I, slightly irritating

 >50%

 15 -30

 Class II, moderately irritating

 25%-50%

 30 -50

 Class III, irritation

 3 %-25%

 >50

 Class IV, very irritating

<3% 

Applicant's summary and conclusion

Interpretation of results:
Category 2B (mildly irritating to eyes) based on GHS criteria
Executive summary:

The test substance was slightly irritating for the eye.